The chronic hyperinsulinemia that accompanies obesity, metabolic syndrome, and type II diabetes mellitus sustains increased hepatic lipogenesis via transcriptional upregulation of lipogenic enzymes. This results in overproduction of VLDL by the liver with resulting accumulation of atherogenic triglyceride-rich lipoproteins in the plasma. Induction of lipogenesis by insulin is mediated by sterol regulatory element binding protein-1c (SREBP-1c) via transcriptional upregulation. SREBP-1c is synthesized as an integral membrane protein localized in the ER where it is associated with its chaperone (SCAP) and regulatory proteins (Insig2, Sec24). We have found that, in addition to promoting transcription of nascent SREBP-1c, insulin also stimulates transport of the SREBP-SCAP complex to the golgi where it undergoes proteolytic cleavage to release the transcriptionally active N-terminal fragment of SREBP-1c (SREBP-1c processing), thereby amplifying the transcriptional effects of insulin. Conversely, cAMP (a surrogate for glucagon) prevents processing of SREBP-1c. Thus, processing of SREBP-1c is a novel and potentially important additional level by which insulin and other factors regulate this pivotal transcription factor. Although the mechanisms by which processing of the cholesterol regulating isoform, SREBP-2, by sterol balance have been extensively studied, little is known about regulation of SREBP-1c processing by insulin and cAMP. We have also found that nascent SREBP-1c is phosphorylated in the presence of insulin via MAPK and PI-3K dependent pathways. Inhibitors of these pathways prevent both phosphorylation of SREBP-1c and insulin induced processing indicating that phosphorylation of SREBP-1c (and possibly of other participating proteins) may mediate the effect of insulin on processing. We therefore propose to delineate the mechanism(s) by which insulin and cAMP regulate proteolytic processing of SREBP-1c by identifying (1) the sites on SREBP-1c that are phosphorylated by insulin, (2) the signaling pathways that mediate the effect of insulin to stimulate and cAMP to inhibit processing, and (3) the functional significance of phosphorylation for regulation of SREBP-1c processing. We will also determine the additional roles of regulation of ER membrane cholesterol content and Insig2 expression by insulin in SREBP-1c processing. These studies will be carried out both in vitro in primary rat hepatocytes and in an in vivo model of hyperinsulinemia, the corpulent JCR:LA-cp rat. Relevance: in obesity, metabolic syndrome, and adult-onset diabetes mellitus, high insulin levels promote formation of fat (triglyceride) by the liver. The resulting elevated levels of triglyceride in the plasma increase the risk of heart attack and stroke. This research examines the role of an important regulatory protein, SREBP-1c in the response of the liver to high insulin levels. The goal of this research is to identify new treatments for hyperlipidemia by understanding the mechanisms by which insulin regulates this protein. [unreadable] [unreadable] [unreadable]